Many Pseudomonas aeruginosa virulence determinants are preferentially expressed during the acute or chronic phases of colonization. For instance, expression of the type III secretion (T3SS) and the cAMP/Vfr signaling (CVS) systems is important for virulence in acute animal infection models. These same factors, however, are not expressed in isolates obtained from cystic fibrosis (CF) patients with chronic P. aeruginosa infections. At least two global regulatory pathways (RsmA and MucA/AlgU) inversely regulate T3SS/CVS and chronic virulence gene expression. The RsmA pathway inversely regulates T3SS/CVS gene expression with biofilm formation (common in the CF airways), and is dependent upon RsmA, an RNA binding protein that regulates gene expression at the post-transcriptional level. The MucA/AlgU pathway activates mucoid conversion (a common trait of CF isolates) but inhibits T3SS/CVS gene expression. Our preliminary data demonstrate that the RsmA and AlgU/MucA regulatory pathways modulate ExsA (the primary transcriptional regulator of the T3SS) and Vfr expression at the post-transcriptional level. The long-term goal of these studies is to define regulatory mechanisms that govern the acute to chronic transition using the T3SS and CVS as model systems by addressing the following specific aims. Aim 1. Determine how RsmA promotes ExsA and Vfr expression at the post-transcriptional level. Our preliminary data indicate that purified RsmA binds directly to the 5' untranslated regions of the vfr and exsA mRNAs, and stimulates Vfr expression in an in vitro translation assay. Based on these data we will test a novel translational derepression model whereby RsmA binding relieves secondary structures within the exsA and vfr mRNAs that sequester the ribosome binding site or otherwise inhibit translation. Aim 2. Determine how the MucA/AlgU system alters the availability of free RsmA to control ExsA and CVS expression. Our data indicate that the expression of RsmA, RsmY, and RsmZ are significantly elevated in the mucA mutant. The experiments proposed in this aim will test the hypothesis that the MucA/AlgU system controls Vfr and ExsA expression by reducing the concentration of free RsmA in cells. Aim 3. Determine how RsmB activity is controlled and how RsmB promotes ExsA and Vfr expression at the post-transcriptional level. Although plasmid-expressed RsmB restores ExsA and Vfr expression in an rsmA,rsmB double mutant, we find that purified RsmB does not bind to the same vfr and exsA mRNA probes bound by RsmA. Purified RsmA and RsmB, however, do bind with similar affinity to a control probe indicating that purified RsmB is active. To determine how RsmB promotes ExsA and Vfr expression we will map the RsmB-responsive region and ask whether RsmB binds to the exsA and vfr mRNAs in vivo.